Electrochemical sensors for cell culture monitoring

ABSTRACT

A device for monitoring a cell culture includes one or more electrochemical sensors configured to be positioned adjacent to or embedded within a medium of a cell culture. The one or more electrochemical sensors are configured to generate signals in accordance with the cell culture. A data storage device is configured to receive and store the signals from the one or more electrochemical sensors. A computation device is configured to analyze the signals from the one or more electrochemical sensors to determine cell activity over time using sensitivity information.

BACKGROUND

Technical Field

The present invention generally relates to cell sensors, and moreparticularly to devices and methods for sensing cell activity usingelectrochemical feedback.

Description of the Related Art

Cells are commonly grown in a lab on dishes or in wells that containnutrients in the form of fluid, gel or solids. These may be human oranimal cells, as well as microorganisms, such as bacteria, fungi,archaea and eukaryotic parasites used in research or clinicaldiagnostics and therapeutics. Cells are typically grown in an incubatorwith controlled temperature and humidity. To determine the degree ofgrowth of cells, the cells are most often monitored visually by a human.For example, samples from potential infection sites in patients areinoculated on a Petri dish and a lab technician may visually inspect thedish after a few hours to a few days to spot signs of bacterial colonyformation. This is labor intensive and also is associated with a riskfor contamination. When cells require a strict anaerobic environment,visually inspecting the cells entails exposing them (even if for alimited period of time) to air, which may delay their growth or lead totheir death.

Visual inspection is widely used to determine bacterial susceptibilityto antibiotics, for example, using a disc-diffusion test, whereinbacterial growth is inhibited around a disc containing an antibioticdrug. However, it is practically impossible for humans to continuouslymonitor cells in a culture. Moreover, for microorganism growth to bevisually evident to the naked eye takes an extremely high number ofcells to be accumulated.

SUMMARY

In accordance with an embodiment of the present invention, a device formonitoring a cell culture includes one or more electrochemical sensorsconfigured to be positioned adjacent to or embedded within a medium of acell culture. The one or more electrochemical sensors are configured togenerate signals in accordance with the cell culture. A data storagedevice is configured to receive and store the signals from the one ormore electrochemical sensors. A computation device is configured toanalyze the signals from the one or more electrochemical sensors todetermine cell activity over time using sensitivity information.

Another device for monitoring a cell culture includes a semiconductorsubstrate having source and drain regions formed therein and a gateconductor being disposed between the source and drain regions to formtransistors that function as electrochemical sensors. A sensing layer isconfigured to interact with a cell culture in a medium. A referenceelectrode disposed within the medium and configured to activate theelectrochemical sensors positioned adjacent to or embedded within themedium of the cell culture, the one or more electrochemical sensorsconfigured to generate signals in accordance with the cell culture. Inone embodiment, the sensing layer includes a gate dielectric, and thegate conductor includes the medium being measured.

A method for monitoring a cell culture includes positioning one or moreelectrochemical sensors adjacent to or embedded within a medium of acell culture; generating signals in accordance with the cell culture;and analyzing the signals from the one or more electrochemical sensorsto determine cell activity over time using sensitivity information.

These and other features and advantages will become apparent from thefollowing detailed description of illustrative embodiments thereof,which is to be read in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The disclosure will provide details in the following description ofpreferred embodiments with reference to the following figures wherein:

FIG. 1 is a block/flow diagram showing a device/method for monitoringelectrochemical signals in a cell culture in accordance with oneembodiment;

FIG. 2 is a block/flow diagram showing a device/method for monitoringelectrochemical signals in a cell culture having a control unit toreceive information from other components in accordance with oneembodiment;

FIG. 3 is a block/flow diagram showing a device/method for monitoringelectrochemical signals in a cell culture having a control unit toreceive information from other components and a reference component forgenerating a control sample in accordance with one embodiment;

FIG. 4 is a diagram showing multiple views of a device for monitoringelectrochemical signals and depicting a sensor array for monitoring acell culture in a Petri dish in accordance with one embodiment;

FIG. 5 is a cross-sectional view of a device for monitoringelectrochemical signals and depicting a microscale electrochemicalsensor in accordance with one embodiment;

FIG. 6 is a cross-sectional view of another device for monitoringelectrochemical signals including a microscale electrochemical sensor inaccordance with one embodiment;

FIG. 7 is a plot showing sensitivity data for pH sensing data inaccordance with one embodiment;

FIG. 8 is a plot showing sensitivity data for Cl⁻ concentration sensingdata in accordance with another embodiment;

FIG. 9 is a cross-sectional view of another device for monitoringelectrochemical signals including a gate dielectric as a sensing surfaceor having a sensing surface thereon and a medium acting as a gateconductor in accordance with one embodiment;

FIG. 10 is a plot showing sensitivity data for pH sensing data inaccordance with one embodiment; and

FIG. 11 is a block/flow diagram showing a system/method for monitoring acell culture in accordance with the present principles.

DETAILED DESCRIPTION

In accordance with the present principles, cell metabolism, viabilityand growth in culture are monitored. In one embodiment, thinelectrochemical sensors are embedded in a culture medium to monitor cellmetabolism viability and growth in culture through measurement of pH,electric current, ion and biomolecule concentration, etc. In a usefulembodiment, an apparatus for monitoring a cell culture includes one ormore electrochemical sensors positioned adjacent to a cell culture orembedded within a cell culture medium, the one or more electrochemicalsensors are capable of recording an electrochemical signal from the cellculture. A power source is provided in electrical communication with theone or more electrochemical sensors. A data storage and computationdevice is configured to receive and analyze the electrochemical signalfrom the one or more electrochemical sensors. A transmitter device maybe provided in electrical communication with the storage and computationdevice so that a pattern of viability, metabolism and growth in the cellculture can be monitored.

In another embodiment, methods for monitoring and analyzing theviability, growth and metabolic activity of cells include recording oneor more signal streams from electrochemical sensors that are adjunct toor embedded within a cell-culture medium and analyzing the signals by acomputerized process to determine different measurements. Themeasurements may include, e.g., intensity of metabolism in the cells,consumption of nutrients from the cell culture medium; rate ofaccumulation of waste products of cell metabolism; temperature; pH;electric current, etc. Sensing may permit for comparison betweendifferent regions within the cell culture and assess dynamics in signalpatterns over time and optionally correct the analysis by comparing thesignals of the cells with reference signals obtained from other cellcultures to provide a corrected analysis.

The methods and systems described herein provide for real timemonitoring of cell cultures. The real-time monitoring enables adetermination of the viability of cells, their metabolic activity, andthe effect of drugs or changing growth conditions on their metabolism orviability. This provides timely detection of bacterial, archaeal andeukaryotic cells (including fungal and parasitic growth), particularlyin clinical samples taken in case of suspected infection, beforecolonies are visible to the naked eye. This also shortens the timeneeded to determine antibiotic susceptibility by detecting change ofgrowth in the presence of different antibiotics at differentconcentrations and enables the monitoring of cells grown in anaerobicconditions without the need to expose cells to air to visually examinethem. In addition, cancer cells can be differentiated from normal cellsby detecting differences in their metabolism or growth pattern, and thesusceptibility of cancer cells to anti-neoplastic treatments (includingchemical and biologic agents or ionizing radiation) in-vitro can beevaluated.

The continuous monitoring and increased sensitivity can be employed tofacilitate academic research by allowing low cost real-time monitoringof growing cells, which can provide indications on the effects ofgenetic and other manipulations on cells or environmental conditions andprovide early detection of cell culture contamination or inappropriateambient environments through unexpected changes in metabolism orviability.

In some embodiments, systems or devices detect electrochemical signalsresulting from metabolic activity in cells grown in culture. The systemsmay include one or more electrochemical sensors, a power source,wireless or wired communication to a digital storage and a computationdevice to analyze data recorded by the electrochemical sensors. Thisinformation is communicated to a user. The electrochemical sensors areplaced adjunct to a surface on which cells are grown or embedded withincell culture medium. Electrochemical sensors provide signal streams fromat least a part of the monitored surface. A sampling frequency ofelectrochemical sensors may vary from continuous to intermittent.Electrochemical sensors may monitor similar surfaces which are cell-free(control) or on which cells grow in a reference setting to correct fornoise. Spatiotemporal changes in signal are recorded and analyzed by acomputer connected to the electrochemical sensors or remotely.

Analysis of signals generated by electrochemical sensors allows foridentification of locations in which signals indicate higher or lowermetabolic activity. When employed on bacterial cell cultures, the systemmay support detection of early stage bacterial growth, estimation of theinoculum, prediction of the type of microorganism(s) growing by thespatial pattern of growth and the growth rate. A similar approach may beapplied to cultures of archaea, fungi, amoeba and other eukaryotic celltypes.

The present principles may be applied to any cell culture for measuringthe consumption of nutrients, generation of cellular waste productsidentification of metabolic responses to interventions (such as exposingcells to a drug, change in nutrients provided, change in ambientconditions or manipulating gene expression), as well as early detectionof malfunction of cells due to a disease (e.g., contamination of theculture or infection with a bacterium, phage, virus or fungus) or toimplausible ambient conditions. Monitoring electrochemical sensors canpotentially add to the ability to differentiate healthy from sicktissues sampled from a human or animal body.

It is to be understood that the present invention will be described interms of a given illustrative architecture; however, otherarchitectures, structures, substrate materials and process features andsteps may be varied within the scope of the present invention.

It will also be understood that when an element such as a layer, regionor substrate is referred to as being “on” or “over” another element, itcan be directly on the other element or intervening elements may also bepresent. In contrast, when an element is referred to as being “directlyon” or “directly over” another element, there are no interveningelements present. It will also be understood that when an element isreferred to as being “connected” or “coupled” to another element, it canbe directly connected or coupled to the other element or interveningelements may be present. In contrast, when an element is referred to asbeing “directly connected” or “directly coupled” to another element,there are no intervening elements present.

The present embodiments may include a design for an integrated circuitchip, which may be created in a graphical computer programming language,and stored in a computer storage medium (such as a disk, tape, physicalhard drive, or virtual hard drive such as in a storage access network).If the designer does not fabricate chips or the photolithographic masksused to fabricate chips, the designer may transmit the resulting designby physical means (e.g., by providing a copy of the storage mediumstoring the design) or electronically (e.g., through the Internet) tosuch entities, directly or indirectly. The stored design is thenconverted into the appropriate format (e.g., GDSII) for the fabricationof photolithographic masks, which typically include multiple copies ofthe chip design in question that are to be formed on a wafer. Thephotolithographic masks are utilized to define areas of the wafer(and/or the layers thereon) to be etched or otherwise processed.

Methods as described herein may be used in the fabrication of integratedcircuit chips. The resulting integrated circuit chips can be distributedby the fabricator in raw wafer form (that is, as a single wafer that hasmultiple unpackaged chips), as a bare die, or in a packaged form. In thelatter case, the chip is mounted in a single chip package (such as aplastic carrier, with leads that are affixed to a motherboard or otherhigher level carrier) or in a multichip package (such as a ceramiccarrier that has either or both surface interconnections or buriedinterconnections). In any case, the chip is then integrated with otherchips, discrete circuit elements, and/or other signal processing devicesas part of either (a) an intermediate product, such as a motherboard, or(b) an end product. The end product can be any product that includesintegrated circuit chips, ranging from toys and other low-endapplications to advanced computer products having a display, a keyboardor other input device, and a central processor.

It should also be understood that material compounds will be describedin terms of listed elements, e.g., SiGe. These compounds includedifferent proportions of the elements within the compound, e.g., SiGeincludes Si_(x)Ge_(1−x) where x is less than or equal to 1, etc. Inaddition, other elements may be included in the compound and stillfunction in accordance with the present principles. The compounds withadditional elements will be referred to herein as alloys.

Reference in the specification to “one embodiment” or “an embodiment” ofthe present principles, as well as other variations thereof, means thata particular feature, structure, characteristic, and so forth describedin connection with the embodiment is included in at least one embodimentof the present principles. Thus, the appearances of the phrase “in oneembodiment” or “in an embodiment”, as well any other variations,appearing in various places throughout the specification are notnecessarily all referring to the same embodiment.

It is to be appreciated that the use of any of the following “/”,“and/or”, and “at least one of”, for example, in the cases of “A/B”, “Aand/or B” and “at least one of A and B”, is intended to encompass theselection of the first listed option (A) only, or the selection of thesecond listed option (B) only, or the selection of both options (A andB). As a further example, in the cases of “A, B, and/or C” and “at leastone of A, B, and C”, such phrasing is intended to encompass theselection of the first listed option (A) only, or the selection of thesecond listed option (B) only, or the selection of the third listedoption (C) only, or the selection of the first and the second listedoptions (A and B) only, or the selection of the first and third listedoptions (A and C) only, or the selection of the second and third listedoptions (B and C) only, or the selection of all three options (A and Band C). This may be extended, as readily apparent by one of ordinaryskill in this and related arts, for as many items listed.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of exampleembodiments. As used herein, the singular forms “a,” “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises,” “comprising,” “includes” and/or “including,” when usedherein, specify the presence of stated features, integers, steps,operations, elements and/or components, but do not preclude the presenceor addition of one or more other features, integers, steps, operations,elements, components and/or groups thereof.

Spatially relative terms, such as “beneath,” “below,” “lower,” “above,”“upper,” and the like, may be used herein for ease of description todescribe one element's or feature's relationship to another element(s)or feature(s) as illustrated in the FIGS. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the FIGS. For example, if the device in theFIGS. is turned over, elements described as “below” or “beneath” otherelements or features would then be oriented “above” the other elementsor features. Thus, the term “below” can encompass both an orientation ofabove and below. The device may be otherwise oriented (rotated 90degrees or at other orientations), and the spatially relativedescriptors used herein may be interpreted accordingly. In addition, itwill also be understood that when a layer is referred to as being“between” two layers, it can be the only layer between the two layers,or one or more intervening layers may also be present.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, these elements should notbe limited by these terms. These terms are only used to distinguish oneelement from another element. Thus, a first element discussed belowcould be termed a second element without departing from the scope of thepresent concept.

Referring now to the drawings in which like numerals represent the sameor similar elements and initially to FIG. 1, an apparatus 100 isillustratively shown for monitoring electrochemical signals in a cellculture. The apparatus 100 includes one or more electrochemical sensors101 positioned adjacent to, on top of, or embedded within a cell culturemedium 102 on which cells are grown. A power source 103 provides powerto the electrochemical sensors 101. The power source 103 may include aportable source, such as a battery or may include a photosensor or otherdevice or may include a plug-in source (for a power grid, etc.). A datastorage and computation device 104 is configured to receive and analyzethe electrochemical signals from the sensors 101. The data storage andcomputation device 104 may be remotely located or locally located but ata different location than the medium 102 and the sensors 101. Atransmitter/receiver device 105 may be connected to the storage andcomputation device 104 to send and receive signals, commands, data, etc.between components, e.g., sensors 101 or to a remote reporting devices,etc.

In one embodiment, the data storage and computation device 104 may beconfigured on a same chip or board or may include separate devices. Inone embodiment, the data storage and computation device 104 includes anapplication specific integrated circuit (ASIC) having a processor andmemory storage. The processor interrogates the sensors 101, controlssample frequency and distributes data measurements from the sensors 101to the memory storage for present or future reference. The memory mayinclude a solid state memory, registers, buffers, etc. for storing thedata measurements from the sensors 101.

The apparatus or devices as described herein may be a system, a method,and/or a computer program product. The computer program product mayinclude a computer readable storage medium (or media) having computerreadable program instructions thereon for causing a processor to carryout aspects of the present invention.

The computer readable storage medium can be a tangible device that canretain and store instructions for use by an instruction executiondevice. The computer readable storage medium may be, for example, but isnot limited to, an electronic storage device, a magnetic storage device,an optical storage device, an electromagnetic storage device, asemiconductor storage device, or any suitable combination of theforegoing. A non-exhaustive list of more specific examples of thecomputer readable storage medium includes the following: a portablecomputer diskette, a hard disk, a random access memory (RAM), aread-only memory (ROM), an erasable programmable read-only memory (EPROMor Flash memory), a static random access memory (SRAM), a portablecompact disc read-only memory (CD-ROM), a digital versatile disk (DVD),a memory stick, a floppy disk, a mechanically encoded device such aspunch-cards or raised structures in a groove having instructionsrecorded thereon, and any suitable combination of the foregoing. Acomputer readable storage medium, as used herein, is not to be construedas being transitory signals per se, such as radio waves or other freelypropagating electromagnetic waves, electromagnetic waves propagatingthrough a waveguide or other transmission media (e.g., light pulsespassing through a fiber-optic cable), or electrical signals transmittedthrough a wire.

Computer readable program instructions described herein can bedownloaded to respective computing/processing devices from a computerreadable storage medium or to an external computer or external storagedevice via a network, for example, the Internet, a local area network, awide area network and/or a wireless network. The network may comprisecopper transmission cables, optical transmission fibers, wirelesstransmission, routers, firewalls, switches, gateway computers and/oredge servers. A network adapter card or network interface in eachcomputing/processing device receives computer readable programinstructions from the network and forwards the computer readable programinstructions for storage in a computer readable storage medium withinthe respective computing/processing device.

Computer readable program instructions for carrying out operations ofthe present invention may be assembler instructions,instruction-set-architecture (ISA) instructions, machine instructions,machine dependent instructions, microcode, firmware instructions,state-setting data, or either source code or object code written in anycombination of one or more programming languages, including an objectoriented programming language such as Smalltalk, C++ or the like, andconventional procedural programming languages, such as the “C”programming language or similar programming languages. The computerreadable program instructions may execute entirely on the user'scomputer, partly on the user's computer, as a stand-alone softwarepackage, partly on the user's computer and partly on a remote computeror entirely on the remote computer or server. In the latter scenario,the remote computer may be connected to the user's computer through anytype of network, including a local area network (LAN) or a wide areanetwork (WAN), or the connection may be made to an external computer(for example, through the Internet using an Internet Service Provider).In some embodiments, electronic circuitry including, for example,programmable logic circuitry, field-programmable gate arrays (FPGA), orprogrammable logic arrays (PLA) may execute the computer readableprogram instructions by utilizing state information of the computerreadable program instructions to personalize the electronic circuitry,in order to perform aspects of the present invention.

Aspects of the present invention are described herein with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems), and computer program products according to embodiments of theinvention. It will be understood that each block of the flowchartillustrations and/or block diagrams, and combinations of blocks in theflowchart illustrations and/or block diagrams, can be implemented bycomputer readable program instructions.

These computer readable program instructions may be provided to aprocessor of a general purpose computer, special purpose computer, orother programmable data processing apparatus to produce a machine, suchthat the instructions, which execute via the processor of the computeror other programmable data processing apparatus, create means forimplementing the functions/acts specified in the flowchart and/or blockdiagram block or blocks. These computer readable program instructionsmay also be stored in a computer readable storage medium that can directa computer, a programmable data processing apparatus, and/or otherdevices to function in a particular manner, such that the computerreadable storage medium having instructions stored therein comprises anarticle of manufacture including instructions which implement aspects ofthe function/act specified in the flowchart and/or block diagram blockor blocks.

The computer readable program instructions may also be loaded onto acomputer, other programmable data processing apparatus, or other deviceto cause a series of operational steps to be performed on the computer,other programmable apparatus or other device to produce a computerimplemented process, such that the instructions which execute on thecomputer, other programmable apparatus, or other device implement thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

The flowchart and block diagrams in the Figures illustrate thearchitecture, functionality, and operation of possible implementationsof systems, methods, and computer program products according to variousembodiments of the present invention. In this regard, each block in theflowchart or block diagrams may represent a module, segment, or portionof instructions, which comprises one or more executable instructions forimplementing the specified logical function(s). In some alternativeimplementations, the functions noted in the blocks may occur out of theorder noted in the figures. For example, two blocks shown in successionmay, in fact, be executed substantially concurrently, or the blocks maysometimes be executed in the reverse order, depending upon thefunctionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts or carry out combinations of special purpose hardwareand computer instructions.

Referring to FIG. 2, in another embodiment, a control unit 107 may becoupled to the apparatus 100 to receive information from components ofthe apparatus 100. The control unit 107 can be any computation device,such as, e.g., a smartphone, laptop, cloud or network service device, acomputer or any other computation device. The control unit 107 canreceive electrochemical signal information from the sensors 101 or fromthe transmit/receive device 105, and can analyze and process theinformation to produce a determination of cell environment, cellviability, metabolism or growth in the cell culture. In someembodiments, the control unit 107 is in communication with the datastorage and computation device 104. In some embodiments, the functionsof transmit/receive device 105 and data storage and computation device104 are incorporated into the control unit 107. In other embodiments,the control unit 107 produces an analysis of metabolic activity of thecell culture over time. The control unit 107 in such an embodimentreceives data from the electrochemical sensors 101, analyzes it, andthen processes it to produce a determination of environmental parametersand measures of metabolic activity in the cell culture.

The control unit 107 may employ its other functionality to alert usersin the vicinity or over a network (e.g., wired, wireless or cellularnetwork) of the conditions or changes to the conditions in the cellcultures. The control unit 107 may include applications (apps) employedfor storing or conveying data (graphs, charts, etc.) or send out warningsignals on the status of the culture (e.g., “dangerous levels ofammonia”). The warning signals may be audible (e.g., phone ringing orphone messaging), text messaged, emailed, etc. The information conveyedmay be simple or extremely complex and may be customized by the user.

Referring to FIG. 3, a reference component 200 may be employed alongwith the apparatus 100 having one or more reference samples 202 in asystem 250. In addition to one or more reference samples 202, thereference component 200 may be in communication with the control unit107. The reference component 200 includes the same components as theapparatus 100 and serves as a model or control to evaluate the data fromthe apparatus 100. The apparatus 100 and the reference component 200, inthis embodiment, may each include an amplifier 206 to boost the signalsfrom the electrochemical sensors 101, 201, respectively. The apparatus100 and the reference component 200 each include transmitter/receiverdevices 105, 205, digital computers 104, 204, and power sources 103,203, configured similarly to each other to monitor their respective cellcultures.

In one embodiment, the system 250 comprises one or more referenceelectrochemical sensors 201 positioned adjacent to a reference sample202. A reference power source 203 is connected with the referencesensors 201 (e.g., infrared sensors and/or other sensors). A referencedata storage and computation device 204 is configured to receive andanalyze the infrared heat signal from the reference infrared sensors201. A reference transmit/receive device 205 is connected with thereference storage and computation device 204 and configured tocommunicate information with the control unit 107.

Referring to FIG. 4, a device 300 for sensing cell activity is shown inaccordance with another illustrative embodiment. A top view 310 of thedevice 300 shows a sensors array 301 and cultures 304. A side view 312shows the device 300 is a Petri dish 305 with a culture medium 303thereon. The device 300 includes a substrate 308 (e.g. a semiconductorwafer, ceramic, printed wiring board, plastic, glass or other suitablematerial) having the sensor array 301 formed thereon. The sensor array301 may include etched metallizations to connect sensors 302 in thesensor array 301 to a power source (on-device or off-device) and datacollection component. See e.g., FIG. 1. The sensor array 301 includes aplurality of individual electrochemical sensors 302 which arepositionable near or embedded within the culture medium 303 and employedto monitor changes in temperature, pH, ion concentration and biomoleculeconcentration, etc. in a cell culture or cultures 304 grown on a Petridish 305 or other container. An electrochemical sensor 302 may include aplurality of microsensors (302), each measuring, e.g., activity and/orsize of a culture mass, e.g., in the range of micrometers, and eachcapable of sensing the presence and concentration of cells.

The sensors 302 and the substrate 308 may be sterile or coated with asterile material or otherwise protected so as to not influence the cellactivity in the medium 303. The sensors 302 may be positioned at regularintervals or may be distributed with different densities depending on arelative position within the Petri dish 305. Each sensor 302 includes aunique signal, an index or address or other distinguishing identifier sothat the position and measurements at that position can be known andstored.

The device 300 may be submerged in the medium 303 or may be positionedover or on the medium 303. In one embodiment, the device 300 is attachedto the Petri dish 305 (e.g., a disposable sticker) and provided as aunit.

Referring to FIG. 5, an electrochemical sensor device 400 is shown inaccordance with one illustrative embodiment. The electrochemical sensordevice 400 includes a substrate 401 (e.g., semiconductor substrate,printed wiring board, etc.). The substrate 401 includes a gatedielectric layer 403 formed thereon. The substrate 401 may include asuitable semiconductor material. In one embodiment, the substrate 401includes a silicon based semiconductor material, e.g., Si, SiGe, SiC,etc. and the dielectric layer 403 may include a silicon oxide, a high-kdielectric or other suitable gate dielectric material. The dielectriclayer 403 may be grown, transferred or deposited on the substrate 401.

Source regions 404 and drain regions 406 are formed within the substrate401. The substrate 401 may include one or more doped wells (not shown)to permit proper transistor device operation. The source regions 404 anddrain regions 406 may be formed by ion implantation, although otherprocesses may be employed. A dielectric layer 412 is formed over thegate dielectric 404. The dielectric layer 412 may include an oxide, anitride, an oxynitride or other suitable dielectric material. Thedielectric layer 412 is patterned to form gate openings through thedielectric layer 412. The patterning process may include a lithographicpatterning process to form an etch mask followed by a reactive ion etchto form the gate openings.

A gate conductor 408 is deposited in the patterned gate openings. Thegate conductor 408 may include, e.g., polycrystalline or amorphoussilicon, germanium, silicon germanium, a metal (e.g., tungsten,titanium, tantalum, ruthenium, zirconium, cobalt, copper, aluminum,lead, platinum, tin, silver, gold), a conducting metallic compoundmaterial (e.g., tantalum nitride, titanium nitride, tungsten silicide,tungsten nitride, ruthenium oxide, cobalt silicide, nickel silicide),carbon nanotube, conductive carbon, graphene, or any suitablecombination of these materials. The conductive material may furthercomprise dopants that are incorporated during or after deposition.

A planarization process may be employed to planarize a top surface andremove excess material from the gate conductor 408 deposition. The gateconductor 408 may be formed using a chemical vapor deposition (CVD)process, although other suitable deposition processes may be employed.The gate conductor 408 may include a planar portion to increase surfacearea of electrochemical sensors 402 formed using the gate conductor 408.A sensing surface 410 is formed over the gate conductor 408 and, inparticular, over a top-most planar portion of the gate conductor 408.

The sensing surface 410 may include a material suitable for contacting asurface of cells, e.g., nutrient broths, agar plates, etc. The sensingsurface 410 includes a coating material having a thin layer of nutrientmedium with cells growing on the medium layer. In other embodiments,cells or materials may be grown in or on solid, gel-like and liquidmedia, which can form the sensing surface 410.

A protection layer 416 may be formed over the dielectric layer 412 withwindows opened up over the sensing surfaces 410. The protection layer416 includes an inert material that does not have an impact on the cellcultures. In one embodiment, the protection layer 416 includes polymericmaterial, such as e.g., polyethelene, polycarbonate, etc.

During operation, a cell medium 414 is employed in contact with thesensing surfaces 410. The cell medium 414 may include any media known inthe art, e.g., employed in Petri dishes, etc. The cell medium may beconductive or non-conductive depending on the application. A referenceelectrode(s) 418 is placed into the medium 414. After a cell culture isintroduced into the medium 414, electrical characteristics are monitoredthrough the medium 414 where cell activity occurs. The changes in themedium 414 due to the cell activity are monitored using a charge orpotential placed on the reference electrode 418. Changes in the mediumand/or due to cell activity change a voltage applied to the gateconductors 408 of the sensors 402. The changes affect the currentpassing between the source and drain regions 404 and 406. This is sensedby sensor circuitry formed in other parts of the substrate 401. Theinformation is conveyed and stored, as described above.

The sensors 402 may be based on field effect transistors technology asdescribed; however, other structures and technology types may beemployed. For example field effect transistor (FET) sensors can bereplaced with or work in conjunction with bipolar junction transistor(BJT) devices with a base connected to the sensing surface 410.

Electrochemical sensors 402 are capable of measuring various parametersincluding the concentration of ions and biomolecules (e.g., lactate,glucose), pH and electric current. These sensors 402 can be micro- andeven nano-scale in size and can also be imprinted on stickers andapplied to various surfaces. The very low price of sensors makes itpractical to use the sensor arrays 402 as disposable products.

The sensing surfaces 410 may be adapted for different measurements andthe array of sensors 402 may include one or more different types ofsensors with different sensing surfaces 410 provided for differentfunctions. For example, non-exhaustive examples for designated sensors402 having unique sensing surface compositions for sensing surfaces 410that are capable of specifically monitoring concentration, activity,etc. may be provided. Illustrative examples include, e.g., a TiN layerfor sensing pH, a AgCl layer to detect Cl−, a gold surface and thiolchemistry for functionalization to detect proteins and miRNA, etc.

In some embodiments, the measurements for each sensor 402 include asensing signal that is represented by a drain current. The sensingsignal may be measured by applying a gate voltage at the referenceelectrode 418, and, in one example, a V_(source)=−V and V_(drain)=30 mVfor an nFET. Other embodiments and outputs are also contemplated.

The sensors 402 for monitoring a cell culture are positioned near to orembedded within the medium 414 of a cell culture for recording aplurality of signals from the cell culture. The sensors 402 monitorsignal streams indicative of ambient conditions, cell viability, growthand metabolism in the cell culture. The signals may include pH,temperature, concentration of sodium, potassium, calcium, chloride,lactate, glucose, electric current, electric potential, etc.

In accordance with the present principles, the ability to continuouslymonitor cells in a culture is provided with minimal human interaction.For example, early detection of changes in a cell culture can bedetermined in accordance with the present principles. The time until aninfection is diagnosed is reduced, and/or the time needed to determinewhat the most effective antibiotic would be to treat a patient isreduced. Cells may stop growing or die due to lack of nutrients,unbalanced ambient conditions or infection by viruses, phages and fungi.Early detection of impaired cell growth is an important consideration inaddressing the problem causing impaired growth. For example, earlydetection may permit for ambient conditions to be modified before cellsexperience irreversible damage. Early detection of an infectionaffecting cells may permit for control measures to be taken to preventspread of the infection to other cells or specimens.

Continuous monitoring of the rate of cell growth may be employed tosupport research related to the use of medications. When medications,change in nutrient supply, environmental changes (e.g., partial pressureof oxygen or carbon dioxide) or other interventions such as geneticmodifications intended or potentially capable of cell growth ormetabolism are investigated, continuous monitoring can quantify theireffect on cells and provide a measure of the kinetics of their action.Impairment in cell growth or metabolic activity, or increased rate ofcell growth may include adverse effects due to drugs. Continuousmonitoring of cell growth may permit for better detection of sucheffects.

Cells are often cultured in small compartments such as wells, which aremore difficult to visually inspect. Spectrometry is employed to measurecell concentration in a liquid medium, but this needs manuallypositioning of wells or other containers in a designated device, whichare not located within the incubator. This is labor intensive, increasesthe risk of mistakes and requires displacement of cells from theirpreferred culture environment, which may have adverse effects on cellgrowth. Automated agar plate inoculation systems exist that performseeding of microbiologic samples on plates and monitor their growthusing a camera but these are not commonly used in the research settingdue to their inflexibility and high price.

The present principles provide automated, low cost cell growthmonitoring, which saves time, reduces labor associated with lab work,saves money lost due to waste of reagents and equipment, promotes theability to get real-time results in cell-related research, and shortenstime to microbiologic results that may affect patient treatment andoutcomes.

Referring to FIG. 6, the electrochemical sensor device 400 is shown inaccordance with another illustrative embodiment. The sensing surface 410may include a material suitable for contacting a surface of cells 420,e.g., nutrient broths, agar plates, etc. The sensing surface 410includes a coating material having a thin layer of nutrient medium withcells 420 growing on the medium layer (e.g., agar) in an aqueous medium422. In other embodiments, cells or materials may be grown in or onsolid, gel-like and liquid media, which can form the sensing surface410.

After a cell culture is introduced into the medium 414, electricalcharacteristics are monitored through the medium 414 where cell activityoccurs. The changes in the medium 414 due to the cell activity aremonitored using a charge or potential placed on the reference electrode418. Changes in the medium and/or due to cell activity change a voltageapplied to the gate conductors 408 of the sensors 402. The changesaffect the current passing between the source and drain regions 404 and406. This is sensed by sensor circuitry formed in other parts of thesubstrate 401. The information is conveyed and stored, as describedabove.

Electrochemical sensors 402 are capable of measuring various parametersincluding the concentration of ions and biomolecules (e.g., lactate,glucose), pH and electric current. These sensors 402 can be micro- andeven nano-scale in size and can also be imprinted on stickers andapplied to various surfaces. The very low price of sensors makes itpractical to use the sensor arrays 402 as disposable products.

The sensing surfaces 410 may be adapted for different measurements andthe array of sensors 402 may include one or more different types ofsensors with different sensing surfaces 410 provided for differentfunctions. For example, non-exhaustive examples for designated sensors402 having unique sensing surface compositions for sensing surfaces 410that are capable of specifically monitoring concentration, activity,etc. may be provided. Illustrative examples include, e.g., a TiN layerfor sensing pH, a AgCl layer to detect Cl−, a gold surface and thiolchemistry for functionalization to detect proteins and miRNA, etc.

An array of chemical FET sensors 402 is embedded in a surface forgrowing cells. The pH can be sensed by making the sensing surface to beTiN, Cl− can be detected by changing the sensing surface to AgCl layer,proteins and miRNA can be detected by using gold surface and thiolchemistry for functionalization. Measurements for each sensor includes asensing signal using the drain current. The sensing signal can bemeasured by applying gate voltage (Vgate) at the reference electrode418, e.g., Vsource=0V and Vdrain=30 mV for nFETs. The sensing surface410 can be of a planar shape or a 3-dimensional shape, e.g., a needleembedded in agar 414. Other embodiments and outputs are alsocontemplated.

Referring to FIG. 7, a plot of drain current (I_(D)) in amperes isplotted against solution voltage (V_(sol)) in Volts to indicate a pHmeasurement by the sensing surfaces 410 (e.g., TiN for measuring pH) anddevices 402. V_(sol) here is the voltage of the reference node 418 (inFIGS. 5 and 6). The pH sensing data measured, in this example, showsplots for pH 5, pH 6 and pH 8 and provides a relationship between the pHdue to cell activity and device related sensitivities (I_(D) changeswith pH changes). The change in threshold voltage (ΔV_(T)), in thisexample, is 42 mV/pH.

Using this information, sensitivity data collected from the sensor 402(FIG. 6) can be employed to correlate measurements to cell activity orstatus. In one example, the pH changes can be determined from electricalsignals from the sensor 410 (e.g., drain current changes, thresholdvoltage changes, etc.).

Referring to FIG. 8, a plot of drain current (I_(D)) in amperes isplotted against gate voltage (V_(G)) in Volts to indicate a Cl−measurement by the sensing surfaces 410 (e.g., AgCl/Ag for measuringCl−) and devices 402. VG here is the voltage of the reference node 418(in FIGS. 5 and 6). The Cl− sensing data measured, in this example,shows plots for 10⁻¹ M KCl (molarity of potassium chloride) to 10⁻⁵ MKCl and provides a relationship between the Cl− concentration due tocell activity and device related sensitivities (I_(D) changes with Cl−concentration).

Using this information, sensitivity data collected from the sensor 402(FIG. 6) can be employed to correlate measurements to cell activity orstatus. In one example, the Cl⁻ concentration changes can be determinedfrom electrical signals from the sensor 410 (e.g., drain currentchanges, etc.).

Referring to FIG. 9, another electrochemical sensor device 440 is shownin accordance with another illustrative embodiment. A sensing surface432 may include a gate dielectric material suitable for contacting cells420, e.g., through nutrient broths, agar plates, etc. The medium 414represents the gate conductor in this structure and the medium 414 is incontact with the gate dielectric or sensing surface 432 (e.g., formed onor of the gate dielectric material).

The sensing surface 432 can include a dielectric material such as, e.g.,HfO₂, Al₂O₃, SiO₂, a dual layer of SiO₂/HfO₂, SiO₂/Al₂O₃, or othersuitable gate dielectric material. The gate dielectric 432 can be formedin a dielectric layer 434 (e.g., SiO₂). The gate dielectric 432 used asis (exposed to agar 414 or cells 420) or an additional bio-layer may bedeposited on top of the gate dielectric to specifically bind targetbio-molecules to form the sensing surface 432. For each sensor 430, asensing signal can include the drain current. In one embodiment, thesensing signal is measured by applying gate voltage (Vgate) at thereference electrode 418. In one example, when Vsource=0V, Vdrain˜30 mVfor nFETs.

After a cell culture is introduced into the medium 414, electricalcharacteristics are monitored through the medium 414 where cell activityoccurs. The changes in the medium 414 due to the cell activity aremonitored using a charge or potential placed on the reference electrode418. Changes in the medium and/or due to cell activity change a voltageapplied to the gate conductors 432 of the sensors 430. The changesaffect the current passing between the source and drain regions 404 and406. This is sensed by sensor circuitry formed in other parts of thesubstrate 401. The information is conveyed and stored, as describedabove.

Electrochemical sensors 430 are capable of measuring various parametersincluding the concentration of ions and biomolecules (e.g., lactate,glucose), pH and electric current. These sensors 430 can be micro- andeven nano-scale in size and can also be imprinted on stickers andapplied to various surfaces. The very low price of sensors makes itpractical to use the sensor arrays 430 as disposable products.

The sensing surfaces 432 may be adapted for different measurements andthe array of sensors 430 may include one or more different types ofsensors with different sensing surfaces 432 provided for differentfunctions. For example, non-exhaustive examples for designated sensors430 having unique sensing surface compositions for sensing surfaces 432that are capable of specifically monitoring concentration, activity,etc. may be provided. While the gate dielectric can be the sensingsurface, the sensing surface may be treated, textured or coated inaccordance with specific applications or measurments. In one embodiment,the sensing layer includes a gate dielectric, and the gate conductorincludes the medium being measured. Other embodiments and outputs arealso contemplated.

Referring to FIG. 10, a plot of drain current (I_(d)) in amperes isplotted against solution voltage (V_(sol)) in Volts to indicate a pHmeasurement by the sensing surfaces 432 (e.g., HfO₂ gate dielectric formeasuring pH) and devices 430. V_(sol) here is the voltage of thereference node 418 (in FIG. 9). The pH sensing data measured, in thisexample, shows plots for pH 5, pH 6, pH 7, pH 8 and pH 10 and provides arelationship between the pH due to cell activity and device relatedsensitivities (I_(d) changes with pH changes).

Referring to FIG. 11, an illustrative method is shown for monitoring acell culture in accordance with the present principles. In block 502,cells are placed (inoculated) into a cell medium, e.g., a Petri dish,well or container. Depending on the measurements to be applied tested ormade, in block 504, one or more drugs or concentrations of drugs areadministered to the cell culture. This is optional depending on the typeof cell culture and the effects being tested or measured. In block 506,an electrochemical sensor array is provided. The sensor array may havebeen present in the container prior to adding the cell medium (e.g., asticker placed in a Petri dish). The sensor array may be placed on topof, near to or in contact with a surface of the cell medium.

In block 508, signal streams are collected from the sensors. This mayinclude monitoring drain currents or monitoring other electricalcharacteristics. In block 510, the signals may be compared to storeddata, to control groups, to other portions of the cell culture. Patternsand other information can be inferred for cell viability, change ingrowth and metabolism, among other things.

Blocks 512, 514 and 516 provide a response to the signals received fromthe sensors. The response depends on the application of the device. Forexample, in block 516, drug susceptibility is detected using the changesmeasured by the sensors. The drug susceptibility is only measured ifdrugs are optionally administered in block 504. In block 514, cell typesmay be detected. This may include determining cancer cells versus normalcells, resistant cells versus non-resistant, etc. In block 512, cellviability or growth is measured or detected. In block 518, based on theresults obtained in blocks 512, 514 and 516, an alert is provided to auser regarding the status or activity of the cell culture. The alert maybe simple (e.g., an alarm) or complex depending on how the system is setup. In one embodiment, a look up table may be stored that indexesconditions with messages that may be employed for sending an alertmessage to a user. The message may be sent to/from a phone, etc.

Having described preferred embodiments for electrochemical sensors forcell culture monitoring (which are intended to be illustrative and notlimiting), it is noted that modifications and variations can be made bypersons skilled in the art in light of the above teachings. It istherefore to be understood that changes may be made in the particularembodiments disclosed which are within the scope of the invention asoutlined by the appended claims. Having thus described aspects of theinvention, with the details and particularity required by the patentlaws, what is claimed and desired protected by Letters Patent is setforth in the appended claims.

What is claimed is:
 1. A device for monitoring a cell culture,comprising: one or more electrochemical sensors configured to bepositioned adjacent to or-embedded within a medium of a cell culture, asensing surface of the one or more electrochemical sensors formeddirectly on a surface of a gate conductor and configured to generatesignals in accordance with cell activity at a region of the cell cultureproximate to the gate conductor of the one or more electrochemicalsensors; a data storage device configured to receive and store thesignals from the one or more electrochemical sensors; and a computationdevice configured to analyze the signals from the one or moreelectrochemical sensors to determine cell activity over time usingsensitivity information.
 2. The device as recited in claim 1, whereinthe data storage device and the computation device are included in asame device and the same device is selected from the group consisting ofa smartphone and a computer.
 3. The device as recited in claim 1,further comprising a transmit device coupled to the one or moreelectrochemical sensors to communicate with at least the computationdevice.
 4. The device as recited in claim 1, wherein the signalsindicate at least one of ambient conditions, cell viability, growth andmetabolism in the cell culture.
 5. The device as recited in claim 1,wherein the signals represent parameters selected from the groupconsisting of: pH, temperature, material concentrations, electriccurrent and electric potential.
 6. The device as recited in claim 5,wherein the material concentrations are selected from the groupconsisting of: sodium, potassium, calcium, chloride, lactate andglucose.
 7. The device as recited in claim 1, further comprising areference component including an electrochemical sensor array to enablea comparison to the one or more electrochemical sensors.
 8. The deviceas recited in claim 1, wherein the device is disposable.
 9. The deviceas recited in claim 1, wherein the one or more electrochemical sensorsinclude transistor devices having a sensing layer formed on a contact ofthe transistor devices.
 10. The device as recited in claim 1, whereinthe computation device is further configured to compare the signals fromdifferent regions of the cell culture.